We wish to understand and describe in molecular detail the mechanisms of transcription, replication, and packaging of the segmented double-stranded RNA chromosomes of the bacterial virus 06. The Specific Aims are focused on expansion of the genetic potential of the 06 system to utilize recombinant DNA technology as a tool to initiate and explore the parameters of DNA RNA replication and packaging. Knowledge of the physical and functional organization of the three genome segments will allow us to use regionally localized strand specific cDNA probes to analyze kinetics of 06 (-) strand synthesis in vivo and to define template requirements and intermediate structures in vivo and in vitro in systems where replication is separated from transcription. Insertion of the 06 procapsid genes (cDNA) encoding P1, P2, P4 and P7, of which two, P1 and P2, have known roles in replication and transcription, into high-expression vectors will allow amplification of the gene products individually for purification and biochemical characterization. Physiological experiments in cells with recombinant plasmids containing two or more of the procapsid genes may reveal specific protein/protein complexes. A 06 replicase assay developed from our current knowledge of 06 RNA termini will be useful in 06 replicase purification and to study the initiation reaction in vitro. Even without this assay the proteins can be purified by conventional means using gel analysis and immunoblot procedures, and used to study binding to ssRNA and dsRNA. Finally, if we are lucky, we will rescue mutant 06 with plasmids carrying an entire 06 cDNA genome segment and we will be able to profitably exploit site specific mutagenic techniques and recover mutants for analysis.